Yasuyuki Nishiyama

A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h‐warts, which was mapped at chromosome 6q24‐25.1. The h‐warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h‐warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h‐warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.

Cloning and characterization of NE-dlg: A novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dlg). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis.

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613‐phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.

Evaluation of Factors Related to Late Recurrence - Later than 10 Years after the Initial Treatment - in Primary Breast Cancer

Background: Breast cancer is associated with a relatively good prognosis. Prognostic factors examined to date are related to early recurrence while those related to late recurrence and their countermeasures remain unclear. Therefore, we examined the factors related to late recurrence. Patients and Methods: From January 1980 to August 2012, 4,774 patients who underwent primary treatment and estrogen (ER) and progesterone receptor (PgR) assessment were enrolled in this study. The patients were divided into two groups, those with a follow-up period <10 years and those without any recurrence at 10 years but who continued follow-up examinations. Recurrence occurred in 711 patients followed up for <10 years and in 51 patients for ≥10 years. Results: The overall 10-year cumulative disease-free survival rate was 79.5%, and the recurrence rate at ≥10 years was 5.8%. A multivariate analysis revealed that the factors related to late recurrence were PgR positivity and positive nodes. This result differed from that for early recurrence in terms of ER/PgR, Ki-67 index and p53 overexpression. Conclusion: PgR positivity and lymph node metastases significantly correlated with late recurrence. Therefore, it is important to evaluate appropriate measures such as treatment period and treatment regimen for hormone-sensitive patients.

Evaluation of an Optimal Cut-Off Point for the Ki-67 Index as a Prognostic Factor in Primary Breast Cancer: A Retrospective Study

The Ki-67 index is an important biomarker for indicating the proliferation of cancer cells and is considered to be an effective prognostic factor for breast cancer. However, a standard cut-off point for the Ki-67 index has not yet been established. Therefore, the aim of this retrospective study was to determine an optimal cut-off point in order to establish it as a more accurate prognostic factor. Immunohistochemical analysis of the Ki-67 index was performed on 4329 patients with primary breast cancer from August 1987 to March 2012. Out of this sample, there were 3186 consecutive cases from September 1997 with simultaneous evaluations of ER, PgR and HER2 status. Coxs proportional hazard model was used to perform univariate and multivariate analyses of the factors related to OS. The hazard ratios (HR) and the p values were then compared to determine the optimal cut-off point for the Ki-67 index. The median Ki-67 index value was 20.5% (mean value 26.2%). The univariate analysis revealed that there was a statistically significant negative correlation with DFS and OS and the multivariate analysis revealed that the Ki-67 index value was a significant factor for DFS and OS. The top seven cut-off points were then carefully chosen based on the results of the univariate analysis using the lowest p-values and the highest HR as the main selection criteria. The multivariate analysis of the factors for OS showed that the cut-off point of 20% had the highest HR in all of the cases. However, the cutoff point of 20% was only a significant factor for OS in the Luminal/HER2- subtype. There was no correlation between the Ki-67 index value and OS in any of the other subtypes. These data indicate that the optimal cut-off point of 20% is the most effective prognostic factor for Luminal/HER2- breast cancer.

The importance of tissue handling of surgically removed breast cancer for an accurate assessment of the Ki-67 index.

Aim Insufficient attention for the Ki-67 immunohistochemistry has been given to the importance of tissue handling for surgical breast cancer specimens. We sought to investigate the effect of fixation status on the Ki-67. Methods We examined the effect of fixative, time to and duration of fixation using surgical specimens, and finally, compared the paired Ki-67 index in the tumour between core needle and surgical specimen. Results The Ki-67 was significantly higher when 10% neutral buffered formalin was used (p=0.0276). Insufficient fixation caused a drastic reduction in the Ki-67 index (p=0.0177), but not significant in oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Sixteen hours delayed time to fixation also caused a reduction of the Ki-67 (p=0.0284), but not significant in ER. Prolonged fixation significantly led to a gradual reduction in the Ki-67 in a time-dependent manner, but not in both ER and HER2. Finally, cutting the tumour before fixation improved fixation status and consequently caused an increased level of the Ki-67 index (p=0.0181), which resulted in a strong correlation of the Ki-67 between core needle and surgical specimen (r=0.8595). Conclusions Tissue handling of surgical specimen is critical for assessing the Ki-67 compared with ER and HER2. We should pay more attention to tissue fixation status for the standard assessment of the Ki-67 index.

Survival time according to the year of recurrence and subtype in recurrent breast cancer.

BACKGROUND Survival for patients with recurrent breast cancer has improved over time due to the introduction of modern systemic therapy. The aim of this study was to determine the impact of subtype and the year of recurrence on the survival times of recurrent breast cancer. METHODS Between 1979 and 2013, 813 patients who underwent initial treatment for primary breast cancer experienced recurrence. They were divided into two groups based on the year of recurrence; before 2000 and after 2001. Survival after recurrence was compared between these groups based on following criteria; subtypes, disease free interval (DFI), and dominant recurrent site. The median follow-up period after recurrence was 4.3 years. RESULTS Survival improved significantly in the after 2001 group, and a significant improvement in survival was only seen in the HER2-enriched subtype. Multivariate analysis revealed that DFI, ER, HER2 status, dominant recurrent site and the Ki-67 index value were significant prognostic factors. In the HER2-enriched subtype, the year of recurrence, DFI and dominant recurrent site were significant independent factors. In the other subtypes, these factors were not correlated with survival. CONCLUSION Our study revealed that the survival rate of patients with only the HER2-enriched subtype significantly improved after recurrence. To prolong the survival time after recurrence of both luminal and triple negative subtypes, the development of novel targeting therapies to overcome refractory recurrent breast cancer is extremely important.

A Comparison of the Hot Spot and the Average Cancer Cell Counting Methods and the Optimal Cutoff Point of the Ki-67 Index for Luminal Type Breast Cancer

Objective: In this case-control study, we investigated the most suitable cell counting area and the optimal cutoff point of the Ki-67 index. Methods: Thirty recurrent cases were selected among hormone receptor (HR)-positive/HER2-negative breast cancer patients. As controls, 90 nonrecurrent cases were randomly selected by allotting 3 controls to each recurrent case based on the following criteria: age, nodal status, tumor size, and adjuvant endocrine therapy alone. Both the hot spot and the average area of the tumor were evaluated on a Ki-67 immunostaining slide. Results: The median Ki-67 index value at the hot spot and average area were 25.0 and 14.5%, respectively. Irrespective of the area counted, the Ki-67 index value was significantly higher in all of the recurrent cases (p < 0.0001). The multivariate analysis revealed that the Ki-67 index value of 20% at the hot spot was the most suitable cutoff point for predicting recurrence. Moreover, higher ΔKi-67 index value (the difference between the hot spot and the average area, ≥10%) and lower progesterone receptor expression (<20%) were significantly correlated with recurrence. Conclusion: A higher Ki-67 index value at the hot spot strongly correlated with recurrence, and the optimal cutoff point was found to be 20%.

Abstract P4-09-26: The difference between metachronous and synchronous bilateral breast cancer in terms of clinical features and biology

Introduction The recent diagnostic modality such as MRI can diagnose a tiny breast lesion even in healthy contralateral breasts. Recently, the rate of contralateral prophylactic mastectomy (CPM) is on the rise in the United States. In Japan, the CPM has been started for selected patients with a high risk. In this study, we divided bilateral breast cancers into the synchronous and the metachronous group and then compared the clinical features and biology. Patients and Methods Out of 216 bilateral breast cancer patients who underwent surgery between 1995 and March 2015, there were 101 synchronous breast cancer cases and 115 metachronous breast cancer cases (interval to the second tumor > 1 year). The items examined were age, tumor size, lymph nodal status, histological type, and biological markers (ER, PgR, HER2, p53 and Ki-67 index values) in the cases with paired data. Results 1. The incidence of metachronous tumors was relatively stable at 2.1–2.7% throughout the period. On the other hand, the incidence of synchronous tumors has increased to 4.4% in the most recent 5-year period and the median interval was 7.1 years. 2. There was no difference in the tumor size of both tumors in the synchronous group, but the second tumor was significantly smaller than the first tumor in the metachronous group (2.2cm to 1.7cm). The node negative rates showed no difference between two groups. 3. The cases with DCIS were seen in 20% and 25% of the synchronous group and 12% and 16% of the metachronous group. Most of the patients (94.3%) with invasive cancer received systemic adjuvant therapy in the metachronous group. 4. The ER positive rates of both tumors were 87.1% and 88.1% in the synchronous group and 71.6% and 68.4% in the metachronous group, respectively. The concordance rates were higher in the synchronous group (p=0.02). Moreover, there was a significant difference in ER positive rates between the two groups (p=0.01) and the ER negative tumors were more frequent in the second tumor of the metachronous group. The PgR negative tumors increased in the second tumor of metachronous cases. The shorter the interval ( 5. The Ki-67 index values significantly increased in the second tumor of the metachronous group, especially in the cases with a shorter interval. However, there was no difference in the synchronous group. The p53 overexpression rates significantly increased in the cases with a shorter interval. 6. The postoperative prognosis for the first tumor did not differ in both groups. Conclusion The incidence of synchronous bilateral breast cancer cases have increased but have remained relatively stable in the metachronous group. The concordance rates of the ER, PgR, Ki-67 and p53 status were higher in the synchronous group but the cases with negative ER, negative PgR, higher Ki-67 values and positive p53 increased in the second tumor of the metachronous group. These findings suggest that adjuvant systemic therapy played a important role in the treatment of bilateral breast cancer but the secondary tumor was more aggressive in the metachronous cases. Citation Format: Arima N, Nishimura R, Osako T, Nishiyama Y, Fujisue M, Okumura Y, Murakami K, Toyozumi Y. The difference between metachronous and synchronous bilateral breast cancer in terms of clinical features and biology. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-26.

Pertuzumab for the treatment of patients with human epidermal growth factor receptor 2‑positive breast cancer in Japan

Pertuzumab, a novel anti-human epidermal growth factor receptor 2 (HER2) agent, is effective for metastatic HER2-positive breast cancer when used in combination with taxane and trastuzumab. The aim of the present study was to describe the use of pertuzumab in Japan. A phase I clinical trial of pertuzumab for HER2-positive metastatic breast cancer was first conducted in the United States in 2001 (study ID no. TOC2297g) and for HER2-positive solid cancers in Japan in 2004 (study ID no. JO17076). However, Japanese patients were not enrolled in a global phase II trial for metastatic breast cancer (study ID no. BO17929) and no phase II trial of pertuzumab for Japanese patients has yet been conducted. A phase III trial on pertuzumab for metastatic breast cancer (CLEOPATRA study), which included 53 Japanese patients, revealed that pertuzumab significantly prolonged progression-free and overall survival. However, the superiority of the pertuzumab group was not verified in the subgroup analysis of Japanese patients, which was not a preplanned analysis. Therefore, a postmarketing clinical trial for Japanese patients with HER2-positive metastatic breast cancer (COMACHI study) was initiated in November, 2013, to investigate the clinical effectiveness of pertuzumab in Japanese patients. As of December, 2014, global trials on pertuzumab in the metastatic and adjuvant settings are currently ongoing. These trials included Japanese patients with HER2-positive breast cancer. Pertuzumab was approved in Japan in August, 2013 due to the positive findings of the CLEOPATRA study. Unlike the United States and Europe, the Japanes Pharmaceutical and Medical Devices Agency approved the administration of pertuzumab as second- or later-line treatment for HER2-positive metastatic breast cancer, as well as first-line treatment. Furthermore, pertuzumab may be used in combination with other chemotherapeutic agents, with the exception of docetaxel. The approval of the expanded use of pertuzumab is likely to accelerate the market penetration of pertuzumab in Japan more quickly compared with other countries.